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Objetivos: O fator de crescimento de hepatócitos (HGF) é um mitógeno secretado por células estromais mesenquimais (MSCs) que regula o crescimento, a motilidade e a morfogênese celular. No microambiente tumoral da medula óssea (MO) (nicho), o HGF interage com o receptor MET nas células leucêmicas ativando vias de sinalização que contribuem para a resistência quimioterápica e, consequentemente, o desenvolvimento de novos compostos que atuam na interação HGF/MET, torna-se crucial para o tratamento da leucemia mieloide aguda (LMA). Assim, investigamos os efeitos de três novos inibidores de MET, B2000, B6000 e C1000 (Brigham University, WO 2011/127192 A2) in vitro e in vivo. Métodos: Células mononucleares da medula óssea humana (BMNCs) de pacientes com LMA e doadores saudáveis (HD) e linhagens leucêmicas (OCI-AML3, MOLM-13) foram tratadas com inibidores de MET e as taxas de apoptose foram avaliadas. O valor de IC50 foi calculado através de resultados de ensaios de viabilidade. A expressão proteica de HGF foi avaliada por imunofluorescência. In vivo, camundongos portadores de LMA agressiva foram tratados com os inibidores de MET (10 mg/kg; 1x/dia; 5 dias) e medula óssea, sangue periférico e baço foram coletados. Um sistema 3D que mimetiza o nicho da MO foi desenvolvido usando uma esponja hemostática para cultura de células estromais mesenquimais (MSCs), células endoteliais da veia umbilical humana (HUVECs) e células leucêmicas. Análises de sinergia foram realizadas usando o pacote Synergy Finder em R. Resultados: Os inibidores promoveram apoptose de BMNCs de pacientes com LMA (p < .01) sem modular células saudáveis. Nas células Molm-13 e OCI-AML3, o tratamento com os compostos resultou em valores baixos de IC50 (< 10 μM). Para validar esses achados em um modelo fisiopatológico, camundongos leucêmicos foram tratados com os inibidores de MET. O composto C1000 demonstrou o efeito mais citotóxico, inibindo a proliferação de células leucêmicas na medula óssea, baço e sangue periférico (p 10). Conclusão: Nossos achados pré-clínicos sugerem que inibidores de MET representam uma classe de agentes terapêuticos com potencial para melhorar significativamente o tratamento de pacientes com LMA. A combinação com Venetoclax pode oferecer uma opção terapêutica mais eficaz e com menor toxicidade, aumentando as chances de cura e melhorando a qualidade de vida dos pacientes. Apoio: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

Aims: Acute lymphoblastic leukemia (ALL), the most prevalent childhood cancer, disrupts the hematopoietic bone marrow (BM) niche, a specialized microenvironment that supports normal hematopoiesis. During ALL development, this niche undergoes remodeling and reprogramming to support tumor growth, hindering blood cell formation. Mesenchymal stromal cells (MSCs), particularly those located in the perivascular niche, play a pivotal role by shielding leukemic lymphoblasts from external stressors, impacting drug responses and potentially contributing to relapse. Understanding this complex interplay between ALL cells and the BM niche is vital for developing effective drug screening models. Therefore, our study aimed to develop an in vitro coculture system that mimics the ALL BM tumor microenvironment. Methods: T-ALL (Jurkat), B-ALL (RS4-11), and MSCs (HS5) cell lines were maintained in RPMI-1640 supplemented with 10% FBS. Tridimensional cocultures were established using hemostatic sponges (Gelfoam, Pfizer) and spheroids (Hanging Drop or Liquid Overlay Technique containing 0, 5, 10 and 20% Matrigel). These cocultures combined normal MSCs (HS5) and ALL cells to mimic different cellular environments within the tumor microenvironment. Different cell ratios (MSC to T-ALL proportion: 1:1, 1:2, 1:5, 2:5) were tested to simulate different cellular environments. To evaluate the system's applicability, the cocultures were treated for 72h with a BCL2 inhibitor, Venetoclax, or an MCL1 inhibitor, AZD5991, as single agents or in combination. Both agents were used at their 10% maximal response concentration (IC10 - 20 nM). Following the treatment, the 3D systems were dissociated using collagenase (for Gelfoam) or trypsin (for spheroids), and cell populations were analyzed by flow cytometry. Results: In comparison to monocultures, T-ALL cell viability was enhanced in cocultures with MSCs in Gelfoam 3D system, with ̃80% of viable ALL cells in all cell ratios tested. Conversely, proliferation of MSCs was diminished compared to monocultures. Although spheroid formation was obtained using hanging drop technique, increasing Matrigel concentrations impaired spheroid formation, except at the highest cell seeding ratio (2:5), which still resulted in highly irregular spheroids with a near-equal distribution of ALL cells and MSCs (1:1). Thus, considering the bone marrow microenvironment, Gelfoam (1:5 cell ratio) was the 3D system that most closely resembled the cellular composition observed in ALL. Cocultures in Gelfoam were treated with Venetoclax and/or AZD5991. The combination therapy effectively inhibited the proliferation of ALL cells in monoculture. However, the combined treatment was not enough to overcome the protective niche action. Importantly, the treatments did not affect MSCs proliferation. Conclusions: Our findings demonstrate an innovative 3D coculture system that successfully replicates the BM stromal niche. This novel platform holds promise for the development of new therapeutic strategies for ALL. Apoio: FAPESP 2017/21801-2, 2019/25247-5, 2021/05320-0; 2023/13850-4; CNPq 303405/2018-0.

Objetivos: Embora a terapia da leucemia mieloide aguda (LMA) tenha registado progressos na última década, a recaída continua a ser o maior desafio terapêutico. As Pim quinases regulam a proliferação e sobrevivência celular e inibem a apoptose ativando vias anti-apoptóticas, como a BCL2, conferindo um pior prognóstico em pacientes com LMA e contribuindo para a recidiva da doença. Portanto, direcionar especificamente as quinases PIM é uma estratégia para o tratamento da LMA. Dessa forma, investigamos os efeitos de um novo inibidor da Pan-PIM quinase, PIM447 (Novartis), em células leucêmicas e exploramos a combinação de PIM447 e Venetoclax, um quimioterápico inibidor de BCL2. Métodos: Células mononucleares (CMs) da medula óssea (MO) de pacientes com LMA e doadores saudáveis (HD) e linhagens de leucemia (OCI-AML3, MOLM-13) foram tratadas com PIM447 e as taxas de apoptose foram avaliadas. A eficácia do PIM447 foi testada em um ensaio organotípico de microscopia, usando CMs de MO de AML cocultivadas com células estromais mesenquimais (MSCs) em meio AIMV-10% FBS e 1,5 mg/mL de colágeno I. Em um modelo xenográfico, camundongos NSG leucêmicos (Molm-13-GFP) foram tratados com PIM447 (2 mg/kg) por 5 dias e medula óssea, sangue periférico e baço foram coletados. A expressão das proteínas BCL2, MCL1 e BCLXL foi avaliada por citometria de. Cocultura de células estromais mesenquimais (MSCs), células endoteliais da veia umbilical humana (HUVECs) e células (Molm-13) foi realizada em um sistema 3D. O pacote Synergy Finder em R foi usado para análises de sinergia. Resultados: O tratamento aumentou em 57% as taxas de apoptose nas CMs de MO leucêmicas (p -10). Em um modelo de nicho da MO, o tratamento com PIM447 inibiu em 70% a proliferação de células Molm-13 (p >,0001) e em 17% blastos de pacientes com LMA (p >,05), sendo mais potente quando combinado com Venetoclax (Molm13: redução de 90%, p < 0001; blastos: redução de 60%). Conclusão: Nossos resultados indicam que o PIM447 possui potente atividade citotóxica contra células leucêmicas, tornando-o um candidato promissor para terapia direcionada em LMA. A combinação sinérgica com um inibidor de BCL2 permite superar a resistência conferida pelo microambiente da medula óssea, abrindo novas perspectivas para o tratamento dessa doença. Apoio: FAPESP 2017/21801-2, 2019/25247-5, 2021/05320-0; CNPq 303405/2018-0.

Objetivos: PIM-quinases, associadas à progressão de cânceres induzidos pelo fator de crescimento de hepatócitos (HGF) por meio de seu receptor, MET, interagem com a serina protease tipo kunitz-2 (SPINT2), no microambiente tumoral (TME) e sua interação pode representar uma importante estratégia terapêutica multialvo na leucemia mieloide aguda (LMA). Analisamos a interação entre SPINT2, HGF e pan-PIM quinases avaliando a expressão gênica e os efeitos de um inibidor de pan-PIM quinase, PIM447 (Novartis), em células leucêmicas e no TME. Métodos: Expressão de mRNA foi obtida do estudo OHSU (2018) e TCGA (cBio Portal). A análise de correlação foi obtida pelo coeficiente de Spearman (r). Células mesenquimais estromais (MSC) da medula óssea (BM) de 5 doadores saudáveis (HD) e 5 pacientes com LMA foram tratadas com 5-Azacitidina (Aza). OCIAML3, MOLM13, U937, RS4;11, Jurkat foram tratadas com PIM447 e as taxas de apoptose foram determinadas. Sangue periférico e células mononucleares (MC) da BM (PBMC/BMMC) de HD foram testados quanto à citotoxicidade. Células leucêmicas foram examinadas para espécies reativas de oxigênio (ROS), produção de óxido nítrico (NO) e potencial de membrana mitocondrial (ΔΨm). Foram realizadas coculturas de células de leucemia com monócitos/macrófagos derivados de monócitos (MDMs) ou MSC com SPINT2 superexpresso. Estatística foi realizada por Kruskal Wallis, ANOVA ou Mann-Whitney (p < 0,05). Resultados: A expressão gênica de 188 pacientes com LMA mostrou que os níveis de HGF correlacionaram-se positivamente com PIM1/PIM2/PIM3 ((r) =,8727;.9094;.9093), p

Objetivos: A leucemia mieloide aguda (LMA) é uma doença sistêmica que continua sendo um grave problema médico em todo o mundo. O acúmulo de células leucêmicas na medula óssea, leva a supressão da hematopoiese normal e promove processos inflamatórios que despertam células-tronco hematopoiéticas quiescentes que proliferam, se diferenciam e geram uma resposta imune desregulada que pode contribuir para a resistência à terapia. Nesse cenário, regular a inflamação pode ser uma estratégia para melhorar as respostas antitumorais. Assim, buscamos compreender o papel dos macrófagos derivados de monócitos no microambiente tumoral da medula óssea (TME) e sua contribuição para a progressão da leucemia, avaliando os efeitos de um composto de tocotrienol derivado da semente de urucum (New Max), uma isoforma da vitamina E. Métodos: Monócitos de células mononucleares de sangue periférico (PBMC) de doadores saudáveis foram isolados de buffy coats. Para ensaios de polarização de macrófagos derivados de monócitos (MDMs), os monócitos foram estimulados com GM-CSF/LPS/INFy ou M-CSF/IL-4 para M1/M2. Células leucêmicas foram adicionadas a uma monocamada de monócitos/macrófagos. Camundongos BALB/c receberam uma única injeção intraperitoneal de LPS para estimular um choque inflamatório e foram tratados com tocotrienol. Análises foram analisadas por citometria de fluxo. Resultados: A análise in vitro mostrou aumento de monócitos clássicos após o tratamento com tocotrienol. Os monócitos tratados com tocotrienol levaram a um aumento da apoptose nas células leucêmicas, sem alterar o microambiente normal. A análise revelou uma população menor de M2-MDMs (HLA-DR-CD206+) e aumento da população de M1-MDMs (HLA-DR+CD80+) (p < 0,0007) em MDMs tratados com tocotrienol. Apoptose de células leucêmicas foi aumentada em coculturas com MDMs tratados com tocotrienol (p < 0,0001). Observou-se aumento da frequência de macrófagos M1 (CD11b+F4/80+CD80+) e diminuição da população M2 (CD11b+F4/80+CD206+) em camundongos tratados com tocotrienol. Conclusão: O tocotrienol foi capaz de ativar o fenótipo de morte de monócitos contra células leucêmicas, sem alterar a sobrevida de monócitos normais, além de ter aumentado a imunidade antitumoral in vitro e in vivo, promovendo a polarização do macrófago M1 e reduzindo o estresse inflamatório com uma ação significativa no TME. Nossos resultados destacam um papel importante do tocotrienol derivado da semente de urucum contra neoplasias hematológicas por meio de uma ação imunológica, sugerindo novos caminhos para estudos futuros. Suporte: FAPESP 2017/21801-2, 2019/25247-5, 2021/05320-0; CNPq303405/2018-0.

Objetivos: Na leucemia mieloide aguda (LMA), MET, um receptor do fator de crescimento de hepatócitos (HGF), contribui para polarização de macrófagos para um fenótipo M2, os quais têm uma ação imunossupressora e interagem com células leucêmicas, promovendo sua sobrevivência, evasão imune e resistência à quimioterapia. Portanto, analisamos a expressão do gene MET em células de pacientes com LMA e avaliar a contribuição de um inibidor específico de MET, B2000 (Brigham University), para a progressão da leucemia com foco no microambiente (TME) inflamatório. Métodos: Expressão de mRNA de MET foram obtidos do estudo TCGA AML, realizado em células isoladas de 290 pacientes com LMA (idade média de 61 anos). Células mononucleares (MC) de sangue periférico (PB) humano e medula óssea (BM) (n = 10) foram obtidas de doadores saudáveis (HD) e usadas para o isolamento de monócitos. OCIAML3, MOLM13 e BMNC de HD e pacientes com LMA (n = 10) foram tratadas com B2000 e as taxas de apoptose foram avaliadas. Para obter macrófagos derivados de monócitos (MDMs), monócitos foram estimulados com GM-CSF/LPS/IFN-y ou M-CSF/IL-4, para polarização M1 ou M2, e usados em coculturas. BALB/c receberam uma injeção intraperitoneal de lipopolissacarídeo (LPS) e tratados com B2000. As análises foram realizadas por citometria de fluxo. Resultados: Observou-se alta expressão de MET em pacientes FAB M1/M3/M7 (p

Objetivos: As neoplasias mieloides são resultantes de alterações moleculares na célula-tronco hematopoiética, sendo a leucemia mieloide aguda (LMA) a mais agressiva e com piores desfechos clínicos. O HCK é um proto-oncogene que codifica uma proteína tirosina-quinase da família Scr, predominantemente expressa em células hematopoiéticas e que possui inibidor farmacológico seletivo. Nos últimos anos houve grandes avanços no entendimento da complexidade molecular das leucemias agudas que tem se traduzido em novas terapias-alvo dirigidas. Desta forma, o objetivo do estudo foi caracterizar a expressão do HCK na mielopoiese normal e maligna, bem como seu impacto sob os desfechos de sobrevida, características clínicas e funções biológicas em pacientes com LMA. Materiais e métodos: Os dados de expressão de HCK em diferentes populações de células hematopoiéticas foram obtidos do estudo GSE24759. Células NB4 e HL-60 foram induzidas a diferenciar com ATRA e a expressão de HCK foi avaliada por PCRq. Os níveis de transcritos de HCK em amostras obtidas de doadores saudáveis e pacientes com neoplasia mieloide (SMD, LMMC, LMC e LMA) foram derivados do banco de dados AmaZonia! 2008. A expressão gênica, dados genéticos e clínicos do estudo LMA TCGA foram obtidos do cBioPortal. Os dados relacionados com tratamentos ex vivo em células de pacientes e linhagens celulares de LMA foram obtidos dos estudos Beat AML e Lee et al. Análises de correlação, associação e comparação entre grupos foram realizados por testes de Spearman, Fisher e Mann-Whitney. A análise de enriquecimento gênico foi realizada por GSEA. Os testes de Log-rank e regressão de Cox foram usados para estimativas de sobrevida. FDR < 0,25 e p valor < 0,05 foram considerados significativos. Resultados: A expressão de HCK é maior nas subpopulações diferenciadas de células mieloides e a indução de diferenciação celular aumenta a expressão de HCK em células NB4 e HL-60 (p < 0,05). A alta expressão de HCK foi observada em pacientes com LMMC, LMC e LMA (p < 0,05). Em pacientes com LMA, os altos níveis de HCK impactou negativamente a sobrevida global e livre de doença (HR > 1; p < 0,05). A alta expressão de HCK foi associada com grupos de piores riscos moleculares e contagem de leucócitos, mas não foi um fator independente de prognóstico. Nas análises de genômica funcional, a alta expressão de HCK foi associada com diversos processos biológicos e moleculares, dos quais destacamos: vias de sinalização PI3K/AKT/mTOR, IL-6/JAK2/STAT3, KRAS, IL2/STAT5 e NOTCH, ROS, hipóxia e resposta inflamatória. Em ensaios ex vivo com amostras de pacientes com LMA e linhagens celulares, destacamos que o HCK esteve associado com sensibilidade e resistência a diversos fármacos de uso clínico, entre eles a azacitidina. Discussão e conclusão: Os dados obtidos demonstram que pacientes com LMA com alta expressão de HCK apresentam um prognóstico desfavorável. A expressão diferencial de HCK está associada a diversas vias de sinalização amplamente relacionadas à leucemogênese, com papel central no crescimento, proliferação e sobrevivência. Nossos dados sugerem que o HCK pode ser um marcador de prognóstico e confirma seu potencial como alvo molecular na LMA. Uma abordagem farmacológica direcionada a essa proteína poderia oferecer uma perspectiva para o desenvolvimento de tratamentos mais eficazes.

Aims: MCL-1 protein is an anti-apoptotic member of BCL-2 protein family. In hematological neoplasms, increased MCL-1 gene expression or protein activity is related to disease relapse, poor prognosis and resistance to chemotherapy, especially to Venetoclax, an BCL-2 inhibitor. The present study aimed at evaluating the MCL-1 gene expression in AML patients as well as investigating the effects of MCL-1 protein inhibition in myeloid cells. Methods: Analysis of MCL-1 gene expression was performed in a cohort of 286 AML patients (OHSU/Nature 2018 study at cbioportal.org). Leukemia myeloid cell lines (U937, KG1a, OCI-AML3) were treated with increasing concentrations of the MCL-1 inhibitor compound AZD5991 (AstraZeneca) for 48, 72 and 96h for determination of the 50% inhibiting concentration (IC50). Apoptosis (annexin-V stain), reactive oxygen species (ROS) production (DFCHA dye) and autophagy (acridine orange dye) were assessed by flow cytometry, after 48h of treatment. Coculture assays were performed by adding leukemia cells onto a monolayer of HS5 mesenchymal stromal cells, followed by treatment with AZD5991 for 48h. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal Wallis tests. p-value < .05 was considered significant. Results: Increased MCL-1 gene expression in AML patients with adverse cytogenetic risk was observed when compared to patients with favorable or intermediate cytogenetic risk (p < .001). All leukemia cell lines showed dose and time-dependent sensitivity with AZD5991. IC50 values of 4.3, 3.4 and 9.2μM in U937; 20.0, 15.2 and 14.9μM in KG1a; and 550, 380 and 420nM in OCI-AML3, for 48, 72 and 96h, respectively. Treatment with AZD5991 (0.5-20μM) in OCI-AML3, U937 and KG1a cells significantly reduced cell viability (69.2%, 50.3%, 40.6%) and increased apoptosis (68.2%, 90.1%, 92.2%) rate in a time and dose-dependent manner, with greater intensity in OCI-AML3 cells. AZD5991 was able to reduce ROS production at 15.1% and 17.2% in U937, 10.3% and 5.6% in KG1a, 59.4% and 45.8% in OCI-AML3, in monoculture and coculture, respectively (p < .05). Similarly, there was a reduction in the formation of acidic vesicles (autophagic process) at 43.0% and 19.0% in U937, 40.5% and 19.0% in KG1a, 62.6% and 18.8% in OCI-AML3, in monoculture and coculture, respectively (p < .05). Increased cell percentage in G0/G1 phases of cell cycle was also observed. Western blot analyses showed decreased MCL-1, Bax and Bak and increased Bim. Discussion and conclusion: MCL-1 overexpression is related with AML progression, and associated with poor prognosis (Li et al. Onco Targets Ther. 2019;12:3295-3304). Our analysis from OHSU data bank showed that patients with adverse cytogenetic risk presented higher MCL-1 gene expression that could induce resistance to pharmacological therapies. Our in vitro studies demonstrated that the MCL1 inhibitor AZD5991 induced apoptosis and reduced viability of leukemia cells, possibly by MCL-1-Bak complex disruption and subsequent Bak-dependent mitochondrial apoptotic pathway activation. The diminished MCL-1 availability in mitochondria, probably caused lower ROS production and impaired autophagy (Mukhopadhyay et al. Apoptosis. 2014;19:555-66). Therefore, these findings provide important tools for further investigations and highlight that MCL-1 inhibitor could be a promising compound for AML treatment due to its potent antitumor activity and may represent a potential therapeutic strategy. Funding: FAPESP e CNPq.

The direct alloresponse, pivotal in transplant rejection, occurs when recipient T cells recognize intact allogeneic peptide-major histocompatibility complex (pMHC) complexes. Despite extensive research, our understanding of alloreactive CD8 + T cells against an individual MHC allele in humans remains limited, especially their precursor frequency, MHC specificity, and peptide specificity. By using K562 cell-based artificial antigen-presenting cells expressing human leukocyte antigen (HLA)-A∗01:01, HLA-A∗02:01, or HLA-A∗03:01, we determined that the precursor frequency of alloreactive CD8 + T cells against a single MHC allele ranges from 0.1% to 0.5%. Further, these cells exhibited MHC specificity regarding proliferation, activation, interferon gamma secretion, and cytolytic ability, with limited crossreactivity toward nontargeted MHC alleles. Focusing on anti-A2 alloreactive CD8 + T cells, we developed a peptide-exchangeable artificial antigen-presenting cell that displays selected peptides on HLA-A∗02:01. From a set of 95 computationally curated A2-restricted peptides most abundant in renal tubular cells, we identified 2 immunogenic kidney peptides across multiple donors. Overall, our findings significantly enhance the understanding of direct alloresponse and provide a toolkit for future mechanistic studies and reproducible patient monitoring., Competing Interests: Declaration of competing interest The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: β-carboline alkaloids exert a distinguished ability to impair cell growth and induce cell death in a variety of cancers and the evaluation of such new therapeutic candidates may denote new possibilities for leukemia treatment. In this present study, we screened 12 β-carboline derivatives containing different substituents at 1- and 3-positions of β-carboline nucleus for their antineoplastic activities in a panel of leukemia cell lines., Methods: The cytotoxic effects of the β-carboline derivatives were evaluated in different leukemia cell lines as well as reactive oxygen species (ROS) generation, autophagy, and important signaling pathways., Results: Treatment with the β-carboline derivatives resulted in a potent antineoplastic activity leading to a reduced cell viability that was associated with increased cell death in a concentration-dependent manner. Interestingly, the treatment of primary mononuclear cells isolated from the peripheral blood of healthy donors with the β-carboline derivatives showed a minor change in cell survival. The antineoplastic activity occurs by blocking ROS production causing consequent interruption of the PI3K/AKT and MAPK/ERK signaling and modulating autophagy processes. Notably, in vivo, AML burden was diminished in peripheral blood and bone marrow of a xenograft mouse model., Conclusions: Our results indicated that β-carboline derivatives have an on-target malignant cell-killing activity and may be promising candidates for treating leukemia cells by disrupting crucial events that promote leukemia expansion and chemotherapy resistance., (© 2024. The Author(s) under exclusive licence to Maj Institute of Pharmacology Polish Academy of Sciences.)

Myeloid neoplasms result from molecular alterations in hematopoietic stem cells, with acute myeloid leukemia (AML) being one of the most aggressive and with a poor prognosis. Hematopoietic cell kinase (HCK) is a proto-oncogene that encodes a protein-tyrosine kinase of the Scr family, and it is highly expressed in AML. The present study investigated HCK expression in normal hematopoietic cells across myeloid differentiation stages and myeloid neoplasm patients. Within the AML cohort, we explored the impact of HCK expression on clinical outcomes and its correlation with clinical, genetic, and laboratory characteristics. Furthermore, we evaluated the association between HCK expression and the response to antineoplastic agents using ex vivo assay data from AML patients. HCK expression is higher in differentiated subpopulations of myeloid cells. High HCK expression was observed in patients with chronic myelomonocytic leukemia, chronic myeloid leukemia, and AML. In patients with AML, high levels of HCK negatively impacted overall and disease-free survival. High HCK expression was also associated with worse molecular risk groups and white blood cell count; however, it was not an independent prognostic factor. In functional genomic analyses, high HCK expression was associated with several biological and molecular processes relevant to leukemogenesis. HCK expression was also associated with sensitivity and resistance to several drugs currently used in the clinic. In conclusion, our analysis confirmed the differential expression of HCK in myeloid neoplasms and its potential association with unfavorable molecular risks in AML. We also provide new insights into HCK biological functions, prognosis, and response to antineoplastic agents., Competing Interests: Conflicts of interest The authors have no competing financial interests., (Copyright © 2023 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier España, S.L.U. All rights reserved.)

The bone marrow (BM) microenvironment (niche) is abnormally altered in acute myeloid leukemia (AML), leading to deficient secretion of proteins, soluble factors, and cytokines by mesenchymal stromal cells (MSC) that modifies the crosstalk between MSC and hematopoietic cells. We focused on a WNT gene/protein family member, WNT5A, which is downregulated in leukemia and correlated with disease progression and poor prognosis. We demonstrated that WNT5A protein upregulated the WNT non-canonical pathway only in leukemic cells, without modulating normal cell behavior. We also introduced a novel WNT5A-mimicking compound, Foxy-5. Our results showed reduction of crucial biological functions that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy, as well as G0/G1 cell cycle arrest. Additionally, Foxy-5 induced early-stage macrophage cell differentiation, a crucial process during leukemia development. At a molecular level, Foxy-5 led to the downregulation of two overexpressed leukemia pathways, PI3K and MAPK, which resulted in a disarrangement of actin polymerization with consequent impairment of CXCL12-induced chemotaxis. Notably, in a novel tri-dimensional bone marrow-mimicking model, Foxy-5 led to reduced leukemia cell growth and similar results were observed in a xenograft in vivo model. Overall, our findings highlight the pivotal role of WNT5A in leukemia and demonstrate that Foxy-5 acts as a specific antineoplastic agent in leukemia, counterbalancing several leukemic oncogenic processes related to the crosstalk in the bone marrow niche, and represents a promising therapeutic option for AML. WNT5A, a WNT gene/protein family member, is naturally secreted by mesenchymal stromal cells and contributes to the maintenance of the bone marrow microenvironment. WNT5A downregulation is correlated with disease progression and poor prognosis. The treatment with Foxy-5, a WNT5A mimetizing compound, counterbalanced several leukemogenic processes that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy and disruption of PI3K and MAPK signaling pathways., (© 2023. The International CCN Society.)

The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. This valuable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms., (© 2023. The International CCN Society.)

We present the case of a 45-year-old man with watery diarrhea for 2 years, leading to marked weight loss (52 kg). On admission, the patient presented with pallor, dehydration and cachexia. Abdominal examination revealed increased bowel sounds, painful and visible intestinal peristalsis, suggesting intestinal obstruction. There was no response to a gluten-free diet and nutritional support. Finally, the patient developed pulmonary infection, septic shock and died 3 months after admission. The diagnosis of CD4+/CD8+ indolent T-cell lymphoma of the GI tract was made post-mortem.

The investigation of tumor microenvironment (TME) is essential to better characterize the complex cellular crosstalk and to identify important immunological phenotypes and biomarkers. The niche is a crucial contributor to neoplasm initiation, maintenance and progression. Therefore, a deeper analysis of tumor surroundings could improve cancer diagnosis, prognosis and assertive treatment. Thus, the WNT family exerts a critical action in tumorigenesis of different types of neoplasms due to dysregulations in the TME. WNT5A, an evolutionary WNT member, is involved in several cellular and physiopathological processes, in addition to tissue homeostasis. The WNT5A protein exerts paradoxical effects while acting as both an oncogene or tumor suppressor by regulating several non-canonical signaling pathways, and consequently interfering in cell growth, cytoskeletal remodeling, migration and invasiveness. This review focuses on a thorough characterization of the role of WNT5A in neoplastic transformation and progression, which may help to understand the prognostic potentiality of WNT5A and its features as a therapeutic target in several cancers. Additionally, we herein summarized novel findings on the mechanisms by which WNT5A might favor tumorigenesis or suppression of cancer progression and discussed the recently developed treatment strategies using WNT5A as a protagonist., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Masson SAS.)

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Blood transfusion is a key intervention for decreasing morbidity and mortality in many cases and, besides its importance, potentially fatal consequences of incompatible transfusion are a great risk to patients. This study evaluated the incidence and specificity of erythrocyte alloantibodies in multi-transfused patients enrolled at an important Regional Blood Center., Materials/methods: This was a single-center retrospective cohort study that eveluated patients enrolled at a Regional Blood Center in a period of four years. A total of 29,128 patient samples were screened, out of which 79 (0.27%) were multiple-transfused patients with alloantibodies identified., Results: The most common alloantibody found was anti-E (22.55%) followed by anti-D (14.71%), anti-C (5.88%), anti-c (5.88%), anti-e (1.96%) and anti-C w (0.98%). We also identified combinations of alloantibodies (25.32%), 5.88% of which showed an IgG autoantibody isolated or combined with alloantibodies. The most frequent reason for the need of blood transfusion included cases of surgery, emergency and urgency (36.71%)., Conclusions: A low rate of development of alloantibodies in multi-transfused patients was found, which could be a consequence of the implementation of red blood cell phenotyping for patients who may receive frequent transfusions, as in the case of some hematological neoplasms and hemoglobinopathies. However, the most common alloantibodies identified were against the Rh and/or Kell systems, with high clinical significance since both can cause delayed hemolytic transfusion reactions. Thus, the implementation of reliable antibody screening tests and the transfusion of phenotyped units for selected patients in all transfusion services represent important measures to increase transfusion safety., (Copyright © 2021 Société française de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved.)

The crosstalk between hematopoietic stem/progenitor cells (HSC), both normal and leukemic, and their neighboring bone marrow (BM) microenvironment (niche) creates a reciprocal dependency, a master regulator of biological process, and chemotherapy resistance. In acute myeloid leukemia (AML), leukemic stem/progenitor cells (LSC) anchored in the protective BM microenvironment, reprogram and transform this niche into a leukemia-supporting and chemoprotective environment. One most important player involved in this crosstalk are CXCL12, produced by the BM mesenchymal stromal cells, and its receptor CXCR4, present onto HSC. The downstream molecular mechanisms involved in CXCL12/CXCR4 axis have many targets, including the Src family members of non-receptor tyrosine kinase (SFK). We herein study the role of one SFK member, the Hematopoietic Cell Kinase (HCK), in CXCL12/CXCR4 pathway and its contribution to the AML pathogenesis. We verified that the inhibition of HCK severely impaired CXCL12-induced migration of leukemic cell lines and CD34 positive cells from AML patients bone marrow, through a disruption of the activation of CXCL12/CXCR4/PI3K/AKT and CXCL12/CXCR4/MAPK/ERK signaling, and by a decreased cytoskeleton dynamic through a lower rate of actin polymerization. We provide new insights into the key role of HCK in conferring a migratory advantage to leukemic cells thought CXCL12/CXCR4 axis. HCK represents an important protein of the main pathway involved in the crosstalk between HSC, and their surrounding milieu. Thus, HCK inhibition could represent a novel approach for the treatment of the acute myeloid leukemia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roversi, Bueno, Pericole and Saad.)

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz-type2 (SPINT2/HAI-2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI-2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5-Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS- and de novo AML-BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI-2 levels up-regulation. Moreover, Aza treatment of HD-BMMSC did not improve SPINT2/HAI-2 levels. To understand the role of SPINT2/HAI-2 down-regulation in BMMSC physiology, SPINT2/HAI-2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS-5 stromal cells and improved survival of CD34 + de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI-2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI-2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down-regulation in SPINT2/HAI-2 gene expression, due to methylation in MDS-BMMSC and de novo AML-BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell-based disorders characterized by ineffective hematopoiesis, increased genomic instability and a tendency to progress toward acute myeloid leukemia (AML). MDS and AML cells present genetic and epigenetic abnormalities and, due to the heterogeneity of these molecular alterations, the current treatment options remain unsatisfactory. Hypomethylating agents (HMA), especially azacitidine, are the mainstay of treatment for high-risk MDS patients and HMA are used in treating elderly AML. The aim of this study was to investigate the potential role of the epigenetic reader bromodomain-containing protein-4 (BRD4) in MDS and AML patients. We identified the upregulation of the short variant BRD4 in MDS and AML patients, which was associated with a worse outcome of MDS. Furthermore, the inhibition of BRD4 in vitro with JQ1 or shRNA induced leukemia cell apoptosis, especially when combined to azacitidine, and triggered the activation of the DNA damage response pathway. JQ1 and AZD6738 (a specific ATR inhibitor) also synergized to induce apoptosis in leukemia cells. Our results indicate that the BRD4-dependent transcriptional program is a defective pathway in MDS and AML pathogenesis and its inhibition induces apoptosis of leukemia cells, which is enhanced in combination with HMA or an ATR inhibitor.

Understanding the molecular basis and mechanisms involved in neoplastic transformation and progression is important for the development of novel selective target therapeutic strategies. Hepatocyte growth factor (HGF)/c-MET signaling plays an important role in cell proliferation, survival, migration and motility of cancer cells. Serine peptidase inhibitor Kunitz type 2 (SPINT2) binds to and inactivates the HGF activator (HGFA), behaving as an HGFA inhibitor (HAI) and impairing the conversion of pro-HGF into bioactive HGF. The scope of the present review is to recapitulate and review the evidence of SPINT2 participation in cancer development and progression, exploring the clinical, biological and functional descriptions of the involvement of this protein in diverse neoplasias. Most studies are in agreement as to the belief that, in a large range of human cancers, the SPINT2 gene promoter is frequently methylated, resulting in the epigenetic silence of this gene. Functional assays indicate that SPINT2 reactivation ameliorates the malignant phenotype, specifically reducing cell viability, migration and invasion in diverse cancer cell lines. In sum, the SPINT2 gene is epigenetically silenced or downregulated in human cancers, altering the balance of HGF activation/inhibition ratio, which contributes to cancer development and progression., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Cross-talk between hematopoietic stem cells (HSCs) and bone marrow stromal cells (BMSCs) is essential for HSCs regulation and leukemogenesis. Studying bone marrow of myelodysplasia patients, a pre-leukemic condition, we found mRNA overexpression of vascular endothelial growth factor A (VEGFA) in CD34 + HSCs and semaphorin 3A (SEMA3A) in BMSCs. To better understand the role of VEGFA and SEMA3A in leukemogenesis, we recruited 30 myelodysplastic syndrome (MDS) patients, 29 acute myeloid leukemia (6 secondary to MDS) patients and 12 controls. We found higher VEGFA expression in de novo AML patients (without prior MDS) group (p=0.0073) and higher SEMA3A expression in all BMSCs patient's samples compared to control group. We then overexpressed VEGFA in an acute myelogenous leukemia cell line, KG1 cells, and in normal CD34 + cells. This overexpression increased KG1 (p=0.045) and CD34 + cell (p=0.042) viability and KG1 (p=0.042) and CD34 + cell (p=0.047) proliferation. Moreover, KG1 and CD34 + cells overexpressing VEGFA also had increased proliferation when co-cultured with human marrow stromal HS5 cells (p=0.045 and p=0.02, respectively). However, co-culture of these transformed cells with HS5 cells overexpressing SEMA3A reduced KG1 (p=0.004) and CD34 + (p=0.009) proliferation. Co-culture of KG1 transformed cells with HS27 cells overexpressing SEMA3A reduced KG1 proliferation as well (p=0.01). To investigate whether the dominant SEMA3A effect over VEGFA could be due to competition for neuropilin1 receptor (NRP1), we performed immunoprecipitation with anti-NRP1 antibody of cell extracts of co-cultured KG1 and HS5 cells, induced or not by VEGFA and SEMA3A recombinant proteins. Results showed a preferential association of NRP1 with SEMA3A, suggesting that SEMA3A can partially reverse the effects caused by the VEGFA preventing its binding with the NRP1 receptor. Since both hematopoietic cells, leukemic and normal, showed similar behavior, we suppose that the attempt to reversion of VEGF effects by SEMA3A is a homeostatic phenomenon in the hematopoietic niche. Finally, we conclude that VEGFA overexpression confers AML cell advantages and SEMA3A may partially reverse this effect; thus, SEMA3A protein combined with VEGFA inhibitors could be beneficial for AML treatment., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aims: Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin-angiotensin system (RAS) may be altered in an animal model of SCD., Main Methods: Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50-75mg/kg/day, 4weeks) treatment on these parameters were also determined., Key Findings: Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart., Significance: Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Myelodysplastic syndromes (MDS) are clonal disorders involving hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, a defective hematopoiesis supporting capacity of mesenchymal stromal cells (MSCs) in the microenvironment niche has been implicated in MDS pathophysiology. The interaction between the dysfunctional MSCs MDS and HSC regulates diverse adhesion-related processes, such as progenitor cell survival, proliferation, differentiation, and self-renewal. As previously reported, a microarray analysis identified serine protease inhibitor kunitz-type 2 (SPINT2), an inhibitor of hepatocyte growth factor (HGF) activation, to be downregulated in MSCs from MDS patients. To define the role of SPINT2 in MDS hematopoietic microenvironment, an analysis of the effect of SPINT2 silencing in MSCs was carried out. We herein reported significantly lower levels of SPINT2 whereas HGF was expressed at higher levels in MSCs from MDS patients compared with healthy controls. SPINT2 underexpression results in an increased expression, production, and secretion of HGF and stromal cell-derived factor 1 (SDF-1) by MSCs. An increased adhesion of normal HSC or malignant cells onto MSCs silenced for SPINT2 was also observed. The altered MSCs adhesion in SPINT2-knockdown cells was correlated with increased CD49b and CD49d expression and with a decrease in CD49e expression. Our results suggest that the SPINT2 underexpression in the MSC from MDS patients is probably involved in the adhesion of progenitors to the bone marrow niche, through an increased HGF and SDF-1 signaling pathway.

Increased γ-globin production and consequent fetal hemoglobin (Hb F, α2γ2) formation is an important modulator of the clinical and hematological features of hemolytic anemias, such as sickle cell disease and β-thalassemia (β-thal). Hb F genes are genetically regulated, but despite numerous studies, the molecular basis of hemoglobin (Hb) switching is not completely understood. Hereditary persistence of fetal Hb (HPFH) is a consequence of impaired switching in adult life, which results in the continued expression of the γ-globin gene. This study was undertaken to identify genes that could be involved in Hb switching and/or maintenance of elevated Hb F levels. Two libraries were constructed using reticulocytes from normal donors and from Brazilian HPFH subjects. Results suggest that the maintenance of Hb F levels could be associated with some gene/protein expression modifications, such as low expression of KLF1, a transcription factor known to contribute to the regulation and modulation of Hb switching, decreased expression of MIER1, known for the recruitment of chromatin remodeling enzymes, and decreased expression of HOOK3. These data suggest new genes that may play a role in globin gene regulation, γ-globin gene expression and augmentation of Hb F levels, and may represent newly-defined cellular pathways for the control of Hb switching in erythroid cells.

Background: Gaucher disease (GD) is the most common glycosphingolipidosis resulting in accumulation of glucoceramide. The most effective treatment for this disease is enzyme replacement therapy (ERT) which involves recombinant enzyme infusion. Enzymatic deficiency in GD patients may induce a cascade of events culminating in secondary effects such as the production of reactive oxygen species (ROS). We investigated the relationship between ROS and GD by analyzing blood oxidative stress markers in GD patients submitted to ERT at different stages during the treatment., Methods: Blood were collected before and just after enzyme infusion. Red blood cell catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and total glutathione (tGSH), and plasma thiobarbituric acid reactive substances (TBARS) were assayed by spectrophotometry. Homocysteine concentrations and related polymorphisms were also studied. Control individuals matched for sex and age were also analyzed., Results: Concentrations of homocysteine and TBARS, and GPx enzyme activity were not different in ERT-treated GD patients. CAT activity was higher while SOD was lower in patients compared to controls. No variations in any of these parameters were found before and just after ERT. Regarding tGSH, a significant increase was observed in GD patients after infusion. Genotypic frequencies studied did not differ from controls or other Brazilian samples., Conclusion: ERT-treated GD patients show an improvement in antioxidant capacity, which is further increased just after recombinant enzyme infusion.